Caracterização estrutural e funcional de metaloproteases isoladas de peçonhas botrópicas
Ano de defesa: | 2013 |
---|---|
Autor(a) principal: | |
Orientador(a): | |
Banca de defesa: | , , , |
Tipo de documento: | Tese |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Universidade Federal de Uberlândia
|
Programa de Pós-Graduação: |
Programa de Pós-graduação em Genética e Bioquímica
|
Departamento: |
Ciências Biológicas
|
País: |
BR
|
Palavras-chave em Português: | |
Palavras-chave em Inglês: | |
Área do conhecimento CNPq: | |
Link de acesso: | https://repositorio.ufu.br/handle/123456789/15738 https://doi.org/10.14393/ufu.te.2013.36 |
Resumo: | CHAPTER II:In the present work, we demonstrate the biochemical and functional characterization of Bothropoidin, a hemorrhagic snake venom metalloproteinase isolated from Bothrops pauloensis snake venom. This protein was purified after three chromatographic steps on CM-Sepharose fast flow, HiTrep Sephacryl S-300 RP and HiTrep Capto Q. Bothropoidin was homogeneous by SDS-PAGE under reducing and nonreducing conditions, and was shown to present a single chain of 49,558 Da by MALDI TOF analysis. The protein presented isoeletric point of 3.76 and the sequence of two fragments obtained by Peptide Mass Fingerprinting (PMF) in MS (MALDI TOF\\TOF) showed significant score when compared with another SVMPs.This enzyme showed proteolytic activity upon azocasein, Aα- chain of fibrinogen, fibrin, collagen and fibronectin. The enzyme was stable at pH between 6-9 and lower temperatures when assayed on azocasein. Moreover, this activity was inhibited by EDTA, 1,10-phenanthroline and β-mercaptoethanol. This enzyme acted on blood clotting altering the aPTT and PT. Bothropoidin was able to induce hemorrhage (MHD=0.98 μg) and necrosis in the gastrocnenius muscles of mice, showing high toxicity. The protein was capable of inhibiting platelet aggregation induced by collagen and ADP and interfered with viability and cell adhesion when incubated with tEnd endothelial cells in a dose dependent manner. Taken together, these results lead us to suggest that Bothropoidin is a hemorrhagic α-fibrinogenase which can contribute significantly to the toxicity of Bothrops pauloensis envenomation. CHAPTER III: A fibrino(geno)lytic nonhemorrhagic metalloproteinase (BleucMP) was purified from Bothrops leucurus snake venom by two chromatographic steps procedure on DEAE-Sephadex A-25 followed by CM-Sepharose Fast Flow column. BleucMP represented 1.75% (w/w) of the crude venom and was homogeneous on SDS-PAGE. BleucMP analyzed by MALDI TOF/TOF, showed a molecular mass of 23,057.54 Da and when alkylated and reduced, the mass is 23,830.40 Da. Their peptides analyzed in MS (MALDI TOF\\TOF) showed significant score when compared with those of other proteins by NCBI-BLAST2 alignment display. As regards their proteolytic activities, BleucMP efficiently acted on fibrinogen, fibrin, and was inhibited by EDTA and 1.10-phenanthroline. This enzyme was also able to decrease significantly the plasma fibrinogen level provoking blood incoagulability, however was devoid of hemorrhagic activity when tested in the mice skin and did not induce relevant biochemical, hematological and histopathological alterations in mice. The aspects addressed in this paper provide data on the effect of BleucMP in envenomation from B. leucurus snakes in order to better understand the effects caused by snake venom metalloproteinase. |
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2016-06-22T18:43:26Z2013-04-082016-06-22T18:43:26Z2013-03-22GOMES, Mário Sérgio Rocha. Caracterização estrutural e funcional de metaloproteases isoladas de peçonhas botrópicas. 2013. 141 f. Tese (Doutorado em Ciências Biológicas) - Universidade Federal de Uberlândia, Uberlândia, 2013. DOI https://doi.org/10.14393/ufu.te.2013.36 DOI https://doi.org/10.14393/ufu.te.2013.36https://repositorio.ufu.br/handle/123456789/15738https://doi.org/10.14393/ufu.te.2013.36CHAPTER II:In the present work, we demonstrate the biochemical and functional characterization of Bothropoidin, a hemorrhagic snake venom metalloproteinase isolated from Bothrops pauloensis snake venom. This protein was purified after three chromatographic steps on CM-Sepharose fast flow, HiTrep Sephacryl S-300 RP and HiTrep Capto Q. Bothropoidin was homogeneous by SDS-PAGE under reducing and nonreducing conditions, and was shown to present a single chain of 49,558 Da by MALDI TOF analysis. The protein presented isoeletric point of 3.76 and the sequence of two fragments obtained by Peptide Mass Fingerprinting (PMF) in MS (MALDI TOF\\TOF) showed significant score when compared with another SVMPs.This enzyme showed proteolytic activity upon azocasein, Aα- chain of fibrinogen, fibrin, collagen and fibronectin. The enzyme was stable at pH between 6-9 and lower temperatures when assayed on azocasein. Moreover, this activity was inhibited by EDTA, 1,10-phenanthroline and β-mercaptoethanol. This enzyme acted on blood clotting altering the aPTT and PT. Bothropoidin was able to induce hemorrhage (MHD=0.98 μg) and necrosis in the gastrocnenius muscles of mice, showing high toxicity. The protein was capable of inhibiting platelet aggregation induced by collagen and ADP and interfered with viability and cell adhesion when incubated with tEnd endothelial cells in a dose dependent manner. Taken together, these results lead us to suggest that Bothropoidin is a hemorrhagic α-fibrinogenase which can contribute significantly to the toxicity of Bothrops pauloensis envenomation. CHAPTER III: A fibrino(geno)lytic nonhemorrhagic metalloproteinase (BleucMP) was purified from Bothrops leucurus snake venom by two chromatographic steps procedure on DEAE-Sephadex A-25 followed by CM-Sepharose Fast Flow column. BleucMP represented 1.75% (w/w) of the crude venom and was homogeneous on SDS-PAGE. BleucMP analyzed by MALDI TOF/TOF, showed a molecular mass of 23,057.54 Da and when alkylated and reduced, the mass is 23,830.40 Da. Their peptides analyzed in MS (MALDI TOF\\TOF) showed significant score when compared with those of other proteins by NCBI-BLAST2 alignment display. As regards their proteolytic activities, BleucMP efficiently acted on fibrinogen, fibrin, and was inhibited by EDTA and 1.10-phenanthroline. This enzyme was also able to decrease significantly the plasma fibrinogen level provoking blood incoagulability, however was devoid of hemorrhagic activity when tested in the mice skin and did not induce relevant biochemical, hematological and histopathological alterations in mice. The aspects addressed in this paper provide data on the effect of BleucMP in envenomation from B. leucurus snakes in order to better understand the effects caused by snake venom metalloproteinase.CAPÍTULO II: No presente trabalho demonstramos a caracterização bioquímica e funcional da Botropoidina, uma metaloprotease hemorrágica isolada da peçonha da serpente Bothrops pauloensis. Esta proteína foi purificada após três passos cromatográficos: CM-Sepharose fast flow, HiTrep Sephacryl S-300 RP, HiTrep Capto Q. Botropoidina mostrou-se homogênea em SDS-PAGE sob condições redutoras e não redutoras e apresentou uma única cadeia polipeptídica de 49.558 Da por análises de MALDI-TOF. A proteína apresentou ponto isoelétrico de 3,76 e a sequência de dois fragmentos obtidos por Peptide Mass Fingerprinting (PMF) em MS (MALDI-TOF\\TOF) apresentou score significativo quando comparado com outras SVMPs. Esta enzima apresentou atividade proteolítica sobre azocaseína, as cadeias Aα e Bβ do fibrinogênio, fibrina, colágeno e fibronectina. A enzima foi estável na faixa de pH de 6-9 e em baixas temperaturas quando ensaiada sobre a azocaseína. Além disso, sua atividade foi inibida por EDTA, 1,10-fenantrolina e β-mercaptoetanol. Esta enzima agiu sobre a coagulação sanguínea alterando os tempos de tromboplastina parcial ativada (TTPa) e de protrombina (TP), além de tornar o sangue de camundongos incoagulável. Botropoidina foi capaz de induzir hemorragia (DMH= 0,98μg) e necrose no músculo gastrocnêmio de camundongos, demonstrando uma alta toxicidade. Esta enzima também foi capaz de inibir a agregação plaquetária induzida por colagéno e ADP e interferiu na adesão e viabilidade de células endoteliais tEnd. Considerados em conjunto, estes resultados nos levam a sugerir que Botropoidina é uma α-fibrinogenase hemorrágica capaz de contribur significativamente para a toxicidade do envenenamento por Bothrops pauloensis. CAPÍTULO III: Neste trabalho, uma metaloprotease não hemorrágica e fibrin(ogen)olitica (BleucMP) foi purificada da peçonha da serpente de Bothrops leucurus em dois passos cromatográficos: DEAE-Sephadex A-25 e CM-Sepharose Fast Flow. BleucMP representa 1,75% (m/m) da peçonha bruta e mostrou-se hemogenea em SDS-PAGE. A análise de BleucMP por MALDI TOF/TOF, reveleu uma massa molecular de 23057,54Da e quando alquilada e reduzida sua massa é de 23830.40Da. A análise dos peptídeos gerados por MS (MALDI TOF/TOF) que mostraram score significantes foi comparado com outras proteínas usando NCBIBLAST2 alignment display. Em relação à atividade proteolítica, BleucMP foi eficiente sobre fibrinogênio, fibrina e azocaseína, e foi inibida por EDTA e 1,10- fenantrolina. Esta enzima também foi capaz de diminuir significativamente os níveis de fibrinogênio plasmático, tornando assim o sangue incoagulável. No entanto foi desprovida de atividade hemorrágica quando testada na pele de camundongos e não induziu relevantes alterações bioquímicas, hematológicas e histopatológicas em camundongos. Os aspectos abordados neste trabalho fornecem dados sobre o efeito da BleucMP no envenenamento de serpentes do gênero Bothrops leucurus, a fim de melhor compreender a ação das metaloproteases no envenenamento causado por serpentes.Doutor em Genética e Bioquímicaapplication/pdfporUniversidade Federal de UberlândiaPrograma de Pós-graduação em Genética e BioquímicaUFUBRCiências BiológicasBothrops pauloensisMetaloprotease hemorrágicaMS(espectrometria de massa)Células tEndAgregação plaquetariaBothrops leucurusVeneno de serpenteMetaloproteaseFibrin(ogen)oliticaBioquímicaSerpente peçonhenta - PeçonhaBothropsHemorrhagic metalloproteinaseMS(mass spectrometry)Platelet aggregationTEnd cellsSnake venomMetalloproteinaseFibrin(ogen)olytic.CNPQ::CIENCIAS BIOLOGICAS::GENETICACaracterização estrutural e funcional de metaloproteases isoladas de peçonhas botrópicasinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisÁvila, Veridiana de Melo Rodrigueshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4720590E9Rodrigues, Renata Santoshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4701853U6Oliveira, Fábio dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4799971D5Clissa, Patrícia Biancahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4728679U1Santos, Juliana Izabel doshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4126683J1http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4774183Y8Gomes, Mário Sérgio Rocha8176339146fda519-77f9-4401-9554-2dfc019dfb92info:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFUinstname:Universidade Federal de Uberlândia (UFU)instacron:UFUTHUMBNAILMarioSergio.pdf.jpgMarioSergio.pdf.jpgGenerated Thumbnailimage/jpeg1414https://repositorio.ufu.br/bitstream/123456789/15738/3/MarioSergio.pdf.jpga3aa36ea4b8330bbdaaf8d64d4e6d413MD53ORIGINALMarioSergio.pdfapplication/pdf6794645https://repositorio.ufu.br/bitstream/123456789/15738/1/MarioSergio.pdf343d3a5f3e6662e344369877202d580bMD51TEXTMarioSergio.pdf.txtMarioSergio.pdf.txtExtracted texttext/plain230986https://repositorio.ufu.br/bitstream/123456789/15738/2/MarioSergio.pdf.txt9540dcc4e71cd82f1c9d78fd394edc32MD52123456789/157382022-08-16 15:37:26.934oai:repositorio.ufu.br:123456789/15738Repositório InstitucionalONGhttp://repositorio.ufu.br/oai/requestdiinf@dirbi.ufu.bropendoar:2022-08-16T18:37:26Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU)false |
dc.title.por.fl_str_mv |
Caracterização estrutural e funcional de metaloproteases isoladas de peçonhas botrópicas |
title |
Caracterização estrutural e funcional de metaloproteases isoladas de peçonhas botrópicas |
spellingShingle |
Caracterização estrutural e funcional de metaloproteases isoladas de peçonhas botrópicas Gomes, Mário Sérgio Rocha Bothrops pauloensis Metaloprotease hemorrágica MS(espectrometria de massa) Células tEnd Agregação plaquetaria Bothrops leucurus Veneno de serpente Metaloprotease Fibrin(ogen)olitica Bioquímica Serpente peçonhenta - Peçonha Bothrops Hemorrhagic metalloproteinase MS(mass spectrometry) Platelet aggregation TEnd cells Snake venom Metalloproteinase Fibrin(ogen)olytic. CNPQ::CIENCIAS BIOLOGICAS::GENETICA |
title_short |
Caracterização estrutural e funcional de metaloproteases isoladas de peçonhas botrópicas |
title_full |
Caracterização estrutural e funcional de metaloproteases isoladas de peçonhas botrópicas |
title_fullStr |
Caracterização estrutural e funcional de metaloproteases isoladas de peçonhas botrópicas |
title_full_unstemmed |
Caracterização estrutural e funcional de metaloproteases isoladas de peçonhas botrópicas |
title_sort |
Caracterização estrutural e funcional de metaloproteases isoladas de peçonhas botrópicas |
author |
Gomes, Mário Sérgio Rocha |
author_facet |
Gomes, Mário Sérgio Rocha |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Ávila, Veridiana de Melo Rodrigues |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4720590E9 |
dc.contributor.referee1.fl_str_mv |
Rodrigues, Renata Santos |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4701853U6 |
dc.contributor.referee2.fl_str_mv |
Oliveira, Fábio de |
dc.contributor.referee2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4799971D5 |
dc.contributor.referee3.fl_str_mv |
Clissa, Patrícia Bianca |
dc.contributor.referee3Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4728679U1 |
dc.contributor.referee4.fl_str_mv |
Santos, Juliana Izabel dos |
dc.contributor.referee4Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4126683J1 |
dc.contributor.authorLattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4774183Y8 |
dc.contributor.author.fl_str_mv |
Gomes, Mário Sérgio Rocha |
contributor_str_mv |
Ávila, Veridiana de Melo Rodrigues Rodrigues, Renata Santos Oliveira, Fábio de Clissa, Patrícia Bianca Santos, Juliana Izabel dos |
dc.subject.por.fl_str_mv |
Bothrops pauloensis Metaloprotease hemorrágica MS(espectrometria de massa) Células tEnd Agregação plaquetaria Bothrops leucurus Veneno de serpente Metaloprotease Fibrin(ogen)olitica Bioquímica Serpente peçonhenta - Peçonha Bothrops |
topic |
Bothrops pauloensis Metaloprotease hemorrágica MS(espectrometria de massa) Células tEnd Agregação plaquetaria Bothrops leucurus Veneno de serpente Metaloprotease Fibrin(ogen)olitica Bioquímica Serpente peçonhenta - Peçonha Bothrops Hemorrhagic metalloproteinase MS(mass spectrometry) Platelet aggregation TEnd cells Snake venom Metalloproteinase Fibrin(ogen)olytic. CNPQ::CIENCIAS BIOLOGICAS::GENETICA |
dc.subject.eng.fl_str_mv |
Hemorrhagic metalloproteinase MS(mass spectrometry) Platelet aggregation TEnd cells Snake venom Metalloproteinase Fibrin(ogen)olytic. |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::GENETICA |
description |
CHAPTER II:In the present work, we demonstrate the biochemical and functional characterization of Bothropoidin, a hemorrhagic snake venom metalloproteinase isolated from Bothrops pauloensis snake venom. This protein was purified after three chromatographic steps on CM-Sepharose fast flow, HiTrep Sephacryl S-300 RP and HiTrep Capto Q. Bothropoidin was homogeneous by SDS-PAGE under reducing and nonreducing conditions, and was shown to present a single chain of 49,558 Da by MALDI TOF analysis. The protein presented isoeletric point of 3.76 and the sequence of two fragments obtained by Peptide Mass Fingerprinting (PMF) in MS (MALDI TOF\\TOF) showed significant score when compared with another SVMPs.This enzyme showed proteolytic activity upon azocasein, Aα- chain of fibrinogen, fibrin, collagen and fibronectin. The enzyme was stable at pH between 6-9 and lower temperatures when assayed on azocasein. Moreover, this activity was inhibited by EDTA, 1,10-phenanthroline and β-mercaptoethanol. This enzyme acted on blood clotting altering the aPTT and PT. Bothropoidin was able to induce hemorrhage (MHD=0.98 μg) and necrosis in the gastrocnenius muscles of mice, showing high toxicity. The protein was capable of inhibiting platelet aggregation induced by collagen and ADP and interfered with viability and cell adhesion when incubated with tEnd endothelial cells in a dose dependent manner. Taken together, these results lead us to suggest that Bothropoidin is a hemorrhagic α-fibrinogenase which can contribute significantly to the toxicity of Bothrops pauloensis envenomation. CHAPTER III: A fibrino(geno)lytic nonhemorrhagic metalloproteinase (BleucMP) was purified from Bothrops leucurus snake venom by two chromatographic steps procedure on DEAE-Sephadex A-25 followed by CM-Sepharose Fast Flow column. BleucMP represented 1.75% (w/w) of the crude venom and was homogeneous on SDS-PAGE. BleucMP analyzed by MALDI TOF/TOF, showed a molecular mass of 23,057.54 Da and when alkylated and reduced, the mass is 23,830.40 Da. Their peptides analyzed in MS (MALDI TOF\\TOF) showed significant score when compared with those of other proteins by NCBI-BLAST2 alignment display. As regards their proteolytic activities, BleucMP efficiently acted on fibrinogen, fibrin, and was inhibited by EDTA and 1.10-phenanthroline. This enzyme was also able to decrease significantly the plasma fibrinogen level provoking blood incoagulability, however was devoid of hemorrhagic activity when tested in the mice skin and did not induce relevant biochemical, hematological and histopathological alterations in mice. The aspects addressed in this paper provide data on the effect of BleucMP in envenomation from B. leucurus snakes in order to better understand the effects caused by snake venom metalloproteinase. |
publishDate |
2013 |
dc.date.available.fl_str_mv |
2013-04-08 2016-06-22T18:43:26Z |
dc.date.issued.fl_str_mv |
2013-03-22 |
dc.date.accessioned.fl_str_mv |
2016-06-22T18:43:26Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
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doctoralThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
GOMES, Mário Sérgio Rocha. Caracterização estrutural e funcional de metaloproteases isoladas de peçonhas botrópicas. 2013. 141 f. Tese (Doutorado em Ciências Biológicas) - Universidade Federal de Uberlândia, Uberlândia, 2013. DOI https://doi.org/10.14393/ufu.te.2013.36 DOI https://doi.org/10.14393/ufu.te.2013.36 |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufu.br/handle/123456789/15738 |
dc.identifier.doi.none.fl_str_mv |
https://doi.org/10.14393/ufu.te.2013.36 |
identifier_str_mv |
GOMES, Mário Sérgio Rocha. Caracterização estrutural e funcional de metaloproteases isoladas de peçonhas botrópicas. 2013. 141 f. Tese (Doutorado em Ciências Biológicas) - Universidade Federal de Uberlândia, Uberlândia, 2013. DOI https://doi.org/10.14393/ufu.te.2013.36 DOI https://doi.org/10.14393/ufu.te.2013.36 |
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https://repositorio.ufu.br/handle/123456789/15738 https://doi.org/10.14393/ufu.te.2013.36 |
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Universidade Federal de Uberlândia |
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UFU |
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Ciências Biológicas |
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Universidade Federal de Uberlândia |
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